Asymmetric cell division has been proposed to be a mechanism by which hematopoietic cells achieve lineage diversity. Cell polarization is a process closely related to asymmetric cell division. The molecular mechanism of these two processes in hematopoietic cells is largely unknown. As part of a long-term goal to study cell fate decision-making in hematopoietic cells, the applicant's laboratory studies the early development of mouse bone marrow hematopoietic progenitor/stem cells (HPSC). The applicant found that when c- Kit? myeloid progenitors and c-Kit??? HPSC were placed in culture with stem cell factor, the ligand of the c-Kit receptor, each mother cell undergoes obligatory asymmetric cell division to generate 2 dissimilar daughter cells, one negative for c-Kit expression and one positive. The c-Kit-negative daughter cell is positive for mature myeloid lineage markers and Numb, a marker of asymmetric cell division, whereas the c-Kit-positive cell is negative for the lineage markers but positive for the Par6 cell polarity complex, microtubule-stabilizing proteins and, in the case of HPSC, other stem cell markers. Capping of c-Kit receptors and polarized redistribution of these protein markers precede their unequal partitioning into the 2 daughter cells during cell division. Activation of the c-Kit receptor is necessary for the generation of the dissimilar daughter cells, and inhibition of c-Kit capping with small-molecule inhibitors blocks polarization and asymmetric cell division. These findings revealed that asymmetric cell division in myeloid progenitors and HPSC can be observed reproducibly in primary cell cultures, with multiple protein markers exhibiting c-Kit-induced polarization and asymmetric segregation. The proposed project will test the following hypothesis: Stimulation of c-Kit signaling leads to capping of c-Kit receptors at an apical pole, local activation of the Par6 polarity machinery, and apical-basal polarization of the microtubule cytoskeleton, thereby giving rise to asymmetric cell division and generation of lineage diversity. The following Specific Aims are proposed: 1) Show that conditional expression, in transgenic mice, of a c-Kit receptor aberrantly targeted to the basal pole will disrupt polarity establishment and interfere with hematopoietic development; 2) Show that conditional expression, in transgenic mice, of a dominant- negative mutant Par6 protein will disrupt polarity establishment and interfere with hematopoietic development. The results of these studies are expected to demonstrate a central role of c-Kit and the Par6 polarity machinery in the polarization and asymmetric cell division of mouse hematopoietic cells, and set the stage for further studies to probe the molecular mechanism of these processes in human hematopoietic cells. Improved understanding of cell polarization and asymmetric cell division in hematopoietic cells should lead to the development of novel techniques to manipulate the cell polarity machinery and direct the differentiation of human blood cells for scientific studies or expand these cells for clinical use. PUBLIC HEALTH RELEVANCE: The project proposes to study how immature blood cells develop distinct domains in the front and back of the cells and how they divide into dissimilar daughter blood cells that each has entirely different cellular properties. Results from this proposed project should help develop new ways to expand blood cells for clinical treatment and find better ways to eradicate leukemia stem cells.